Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Members of the GH3 Family of Proteins Conjugate 2,4-D and Dicamba with Aspartate and Glutamate.

Auxin homeostasis is a highly regulated process that must be maintained to allow auxin to exert critical growth and developmental controls. Auxin conjugase and hydrolase family proteins play important roles in auxin homeostasis through means of storage, activation, inactivation, response inhibition and degradation of auxins in plants. We systematically evaluated 60 GRETCHEN HAGEN3 (GH3) proteins from diverse plant species for amino acid conjugation activity with the known substrates jasmonic acid (JA), IAA and 4-hydroxybenzoate (4-HBA). While our results largely confirm that Group II conjugases prefer IAA, we observed no clear substrate preference among Group III proteins, and only three of 11 Group I proteins showed the expected preference for JA, indicating that sequence similarity does not always predict substrate specificity. Such a sequence-substrate relationship held true when sequence similarity at the acyl acid-binding site was used for grouping. Several GH3 proteins could catalyze formation of the potentially degradation-destined aspartate (Asp) and glutamate (Glu) conjugates of IAA and the synthetic auxins 2,4-D and dicamba. We found that 2,4-D-Asp/Glu conjugates, but not dicamba and IAA conjugates, were hydrolyzed in Arabidopsis and soybean by AtILL5- and AtIAR3-like amidohydrolases, releasing free 2,4-D in plant cells when conjugates were exogenously applied to seedlings. Dicamba-Asp or dicamba-Glu conjugates were not hydrolyzed in vivo in infiltrated plants nor in vitro with recombinant amidohydrolases. These findings could open the door for exploration of a dicamba herbicide tolerance strategy through conjugation.

Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.

Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants.

Ablation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve sufficient specificity of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter specificity to be used to enhance targeting specificity. Using a transient system, we first show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.